From Thermus Thermophilus Hb27 Malate Dehydrogenase Gene Cloning And Expression, Enzyme Inhibition Kinetics And Protein Folding Research

Malate dehydrogenase(MDH,EC 1.1.1.37)is an essential metabolic enzyme in tricarboxylic acid cycle.It catalyzes reversible oxidation of L-malate to oxaloacetate with the concomitant reduction of NAD⁺. For its highly selectivity of 4-carbon dicarboxylic acid substrate, it can be used as biocatalysts in the production of chiral 2-hydroxyacid,which is important in pharmaceutical industry. Moreover, MDH has homo-dimer structure, which is often used as a model to study oligomeric protein structure and function relationship. In this research, we have cloned the full-length sequence encoding Tt-MDH gene from the genomic DNA of Thermus thermophilus HB27 and expressed the active and soluble recombinant Tt-MDH successfully in E. coli. After a simple purification procedure, we obtained this thermostable enzyme, then characterized its biochemical and unfolding properties in detail in the following study.The effects of pH, temperature and ions on the activity of Tt-MDH were studied when using L-malate and NAD⁺ as the substrates. The results showed a optimum pH range of 11.5 and optimum temperature of 80℃. These results suggested that Tt-MDH might play important physiological roles in vivo to adapt Thermus thermophilus HB27 to the extreme temperatures and specific nutritional conditions.Highly purified Tt-MDH was studied for its inhibition kinetics during GdnHCl denaturation. We found that GdnHCl inactivated the activity of Tt-MDH in a dose dependent manner. The IC₅₀ value was 1.45 mol The time-interval kinetic studies shown that the inactivation followed first-order reaction kinetics with a biphasic process. The spectroflurorimetry results showed that GdnHCl conspicuously induced the tertiary structural change of Tt-MDH with exposure of its hydrophobic surfaces.In conclusion, Our this study is useful to understand its heat-resistant mechanism, folding mechanism, structure and function of this new thermostable Tt-MDH, as well as the construct of high efficient and heat-tolerant engineering strains and their possible applications in industry. In addition, it is meaningful to develop the drugs with Tt-MDH as the targets in order to treat human diseases with relation to Tt-MDH with the inhibitors of Tt-MDH.